Raman microspectroscopy enables one to acquire spectra of Raman scattering with a spatial resolution in the order of a few μm3 and thus to study the natural composition of biological objects such as tissues, single cells and cellular organelles in a non-invasive way. In this work, we used Raman microspectroscopy to investigate vacuoles of the opportunistic human yeast pathogen Candida albicans. Large sets of Raman spectra of vacuoles were collected based on different cultivation protocols. The sets of the spectra were evaluated using the multivariate statistical method of singular value decomposition. Based on the spectral analysis, we characterized the chemical composition of the vacuoles. We found out that the vacuoles of cells cultured differently or in different media vary particularly in the concentration of polyphosphate, represented in the spectra by the peak near 1155 cm-1 . Interestingly, the wavenumber position of the polyphosphate peak may also be shifted by several cm-1 . We studied these shifts in vitro with sodium hexametaphosphate as a model of vacuolar polyphosphate. Based on these experiments, we suggest that the peak position is significantly influenced by the concentration of divalent cations.

Title: Raman microspectroscopy of living cells and biological tissues Author: Šárka Moudříková Department / Institute: Institute of Physics of Charles University Supervisor of the doctoral thesis: doc. RNDr. Peter Mojzeš, CSc., Institute of Physics of Charles University Abstract: Raman microscopy combines Raman spectroscopy with optical confocal microscopy and thus provides information on chemical composition of a sample with a µm3 resolution. In this thesis, Raman microscopy has been used to study microalgae-unicellular photosynthetic organisms that are greatly relevant for the Earth's environment as well as for biotechnological applications. Raman microscopy of photosynthetic organisms struggles with a highly intensive background of the spectra, which is formed by fluorescence of cellular photosynthetic apparatus. In this thesis, we have developed a fast and reliable photobleaching method that suppresses the unwanted background; this method has enabled us to study intracellular distribution of algal biomolecules such as proteins, starch, lipids and polyphosphate. We have investigated an evolution of these structures during a cell cycle of a model microalga Desmodesmus quadricauda. Next, we have developed a method for quantitative analysis of polyphosphate in a cellular culture of a microalga Chlorella...